REAL-TIME PCR WITH MOLECULAR BEACONS

Real-time PCR was originally developed for use with TaqMan probe system. The replacement of TaqMan probes with molecular beacon probes represents a major advance in real-time PCR assays. Molecular beacons are single-stranded nucleic acid molecules that possess a stem-and-loop structure. The loop portion of the molecule serves as a probe sequence that is complementary to a target nucleic acid. The stem is formed by the annealing of two complementary arm sequences that are on either side of the probe sequence. The arm sequences are unrelated to the target. A fluorescent moiety is attached to the end of one arm and a non-fluorescent quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, consequently disabling the fluorophore. When the probe encounters a target molecule, it forms a hybrid that is stronger and more stable than the hybrid formed by the arm sequences. The probe then undergoes a spontaneous conformational change that forces the arm sequences apart, separating the fluorophore from the quencher. The fluorophore is now able to fluoresce when illuminated by blue light. Molecular beacons are thermostable and do not prime or interfere with PCR reactions. This makes it possible to include target-specific molecular beacons in a PCR reaction mix, and to use them as reporter probes in real-time PCR reactions. PCR is carried out in the ABI 7700 prism, which measures the fluorescence generated by the molecular beacon-target hybrids at the annealing step of each PCR cycle. The instruments to be purchased through this proposal will be used to construct, perform, and analyze real-time polymerase chain reaction (PCR) and real-time reverse transcription (RT) PCR assays. The requested instrument package includes an oligonucleotide synthesizer suited for multi-color molecular beacon synthesis, an HPLC apparatus for molecular beacon purification, and the equipment required for real-time PCR assays. A group of diverse projects will be pursued including: i) the analysis of M tuberculosis virulence and drug-resistance genes, ii) the regulation of tuberculosis granulomas, iii) regulation of adenovirus gene expression, iv) characterizing genetic polymorphisms in cancer, v) analyzing host and bacterial gene expression in Staphylcoccal infections, vi) characterizing genetic polymorphisms in human genetic diseases, and vii) development of a novel rapid diagnostic test for determination of drug resistance in tuberculosis.